Mouse and human resistins impair glucose transport in primary mouse cardiomyocytes, and oligomerization is required for this biological action.

The adipocytokine resistin impairs glucose tolerance and insulin sensitivity in rodents. Here, we examined the effect of resistin on glucose uptake in isolated adult mouse cardiomyocytes. Murine resistin reduced insulin-stimulated glucose uptake, establishing the heart as a resistin target tissue. Notably, human resistin also impaired insulin action in mouse cardiomyocytes, providing the first evidence that human and mouse resistin homologs have similar functions. Resistin is a cysteine-rich molecule that circulates as a multimer of a dimeric form dependent upon a single intermolecular disulfide bond, which, in the mouse, involves Cys26; mutation of this residue to alanine (C26A) produces a monomeric molecule that appears to be bioactive in the liver. Remarkably, unlike native resistin, monomeric C26A resistin had no effect on basal or insulin-stimulated glucose uptake in mouse cardiomyocytes. Resistin impairs glucose uptake in cardiomyocytes by mechanisms that involve altered vesicle trafficking. Thus, in cardiomyocytes, both mouse and human resistins directly impair glucose transport; and in contrast to effects on the liver, these actions of resistin require oligomerization.

The intrinsic circadian clock within the cardiomyocyte.

Circadian clocks are intracellular molecular mechanisms that allow the cell to anticipate the time of day. We have previously reported that the intact rat heart expresses the major components of the circadian clock, of which its rhythmic expression in vivo is consistent with the operation of a fully functional clock mechanism. The present study exposes oscillations of circadian clock genes [brain and arylhydrocarbon receptor nuclear translocator-like protein 1 (bmal1), reverse strand of the c-erbaalpha gene (rev-erbaalpha), period 2 (per2), albumin D-element binding protein (dbp)] for isolated adult rat cardiomyocytes in culture. Acute (2 h) and/or chronic (continuous) treatment of cardiomyocytes with FCS (50% and 2.5%, respectively) results in rhythmic expression of circadian clock genes with periodicities of 20-24 h. In contrast, cardiomyocytes cultured in the absence of serum exhibit dramatically dampened oscillations in bmal1 and dbp only. Zeitgebers (timekeepers) are factors that influence the timing of the circadian clock. Glucose, which has been previously shown to reactivate circadian clock gene oscillations in fibroblasts, has no effect on the expression of circadian clock genes in adult rat cardiomyocytes, either in the absence or presence of serum. Exposure of adult rat cardiomyocytes to the sympathetic neurotransmitter norephinephrine (10 microM) for 2 h reinitiates rhythmic expression of circadian clock genes in a serum-independent manner. Oscillations in circadian clock genes were associated with 24-h oscillations in the metabolic genes pyruvate dehydrogenase kinase 4 (pdk4) and uncoupling protein 3 (ucp3). In conclusion, these data suggest that the circadian clock operates within the myocytes of the heart and that this molecular mechanism persists under standard cell culture conditions (i.e., 2.5% serum). Furthermore, our data suggest that norepinephrine, unlike glucose, influences the timing of the circadian clock within the heart and that the circadian clock may be a novel mechanism regulating myocardial metabolism.

Evidence for mitochondrial thioesterase 1 as a peroxisome proliferator-activated receptor-alpha-regulated gene in cardiac and skeletal muscle.

The physiological role of mitochondrial thioesterase 1 (MTE1) is unknown. It was proposed that MTE1 promotes fatty acid (FA) oxidation (FAO) by acting in concert with uncoupling protein (UCP)3. We previously showed that ucp3 is a peroxisome proliferator-activated receptor-alpha (PPAR alpha)-regulated gene, allowing induction when FA availability increases. On the assumption that UCP3 and MTE1 act in partnership to increase FAO, we hypothesized that mte1 is also a PPAR alpha-regulated gene in cardiac and skeletal muscle. Using real-time RT-PCR, we characterized mte1 gene expression in rat heart and soleus muscles. Messenger RNA encoding for mte1 was 3.2-fold higher in heart than in soleus muscle. Cardiac mte1 mRNA exhibited modest diurnal variation, with 1.4-fold higher levels during dark phase. In contrast, skeletal muscle mte1 mRNA remained relatively constant over the course of the day. High-fat feeding, fasting, and streptozotocin-induced diabetes, interventions that increase FA availability, muscle PPAR alpha activity, and muscle FAO rates, increased mte1 mRNA in heart and soleus muscle. Conversely, pressure overload and hypoxia, interventions that decrease cardiac PPAR alpha activity and FAO rates, repressed cardiac mte1 expression. Specific activation of PPAR alpha in vivo through WY-14643 administration rapidly induced mte1 mRNA in cardiac and skeletal muscle. WY-14643 also induced mte1 mRNA in isolated adult rat cardiomyocytes dose dependently. Expression of mte1 was markedly lower in hearts and soleus muscles isolated from PPAR alpha-null mice. Alterations in cardiac and skeletal muscle ucp3 expression mirrored that of mte1 in all models investigated. In conclusion, mte1, like ucp3, is a PPAR alpha-regulated gene in cardiac and skeletal muscle.

Regulation of insulin-responsive aminopeptidase expression and targeting in the insulin-responsive vesicle compartment of glucose transporter isoform 4-deficient cardiomyocytes.

In adipocytes and cardiac or skeletal muscle, glucose transporter isoform 4 (GLUT4) is targeted to insulin-responsive intracellular membrane vesicles (IRVs) that contain several membrane proteins, including insulin-responsive aminopeptidase (IRAP) that completely colocalizes with GLUT4 in basal and insulin-treated cells. Cardiac GLUT4 content is reduced by 65-85% in IRAP knockout mice, suggesting that IRAP may regulate the targeting or degradation of GLUT4. To determine whether GLUT4 is required for maintenance of IRAP content within IRVs, we studied the expression and cellular localization of IRAP and other GLUT4 vesicle-associated proteins, in hearts of mice with cardiac-specific deletion of GLUT4 (G4H-/-). In G4H-/- hearts, IRAP content was reduced by 60%, but the expression of other vesicle-associated proteins, namely cellugyrin, IGF-II/mannose-6-phosphate, and transferrin receptors, secretory carrier-associated membrane proteins and vesicle-associated membrane protein were unchanged. Using sucrose gradient centrifugation and cell surface biotinylation, we found that IRAP content in 50-80S vesicles where GLUT4 vesicles normally sediment was markedly depleted in G4H-/- hearts, and the remaining IRAP was found in the heavy membrane fraction. Although insulin caused a discernible increase in cell surface IRAP content of G4H-/- cardiomyocytes, cell surface IRAP remained 70% lower than insulin-stimulated controls. Immunoabsorption of intracellular vesicles with anticellugyrin antibodies revealed that IRAP content was reduced by 70% in both cellugyrin-positive and cellugyrin-negative vesicles. Endosomal recycling, as measured by transferrin receptor recycling was normal. Thus, GLUT4 and IRAP content of early endosome-derived sorting vesicles and of IRVs are coordinately regulated, and both proteins are required for maintenance of key constituents of these compartments in cardiac muscle cells in vivo.

Insulin signaling coordinately regulates cardiac size, metabolism, and contractile protein isoform expression.

To investigate the role of insulin signaling on postnatal cardiac development, physiology, and cardiac metabolism, we generated mice with a cardiomyocyte-selective insulin receptor knockout (CIRKO) using cre/loxP recombination. Hearts of CIRKO mice were reduced in size by 20-30% due to reduced cardiomyocyte size and had persistent expression of the fetal beta-myosin heavy chain isoform. In CIRKO hearts, glucose transporter 1 (GLUT1) expression was reduced by about 50%, but there was a twofold increase in GLUT4 expression as well as increased rates of cardiac glucose uptake in vivo and increased glycolysis in isolated working hearts. Fatty acid oxidation rates were diminished as a result of reduced expression of enzymes that catalyze mitochondrial beta-oxidation. Although basal rates of glucose oxidation were reduced, insulin unexpectedly stimulated glucose oxidation and glycogenolysis in CIRKO hearts. Cardiac performance in vivo and in isolated hearts was mildly impaired. Thus, insulin signaling plays an important developmental role in regulating postnatal cardiac size, myosin isoform expression, and the switching of cardiac substrate utilization from glucose to fatty acids. Insulin may also modulate cardiac myocyte metabolism through paracrine mechanisms by activating insulin receptors in other cell types within the heart.